Production, one-step purification, and characterization of a keratinolytic protease from Serratia marcescens P3

摘要

Applications of keratinases demand efficient production and downstream processing. This work reports the production, purification and partial characterization of a keratinolytic protease from Serratia marcescens P3. The influence of temperature, initial pH and feather meal concentration on enzyme production was investigated using response surface methodology. Maximal protease production (12.5UmL~(-1)) was observed at 18-30℃, 10-24gL~(-1) feather meal, and pH values of 6.5-8.5. The enzyme was recovered from culture supernatants using aqueous two-phase systems (ATPSs), and higher yields (68%) were obtained in polyethylene glycol (PEG) 4000-sodium citrate and PEG 4000-ammonium sulfate systems. These ATPS were effective for enzyme purification, resulting in an electrophoretically homogeneous protein of 53 kDa. Sequencing of tryptic peptides identified this enzyme as a metallopro-tease belonging to the serralysin family of metzincins. The purified enzyme showed optimal activity at 40-45 ℃ and pH 6.5, being also active toward azokeratin.